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- EMDB-26477: VchTnsC AAA+ with DNA (double heptamer) -

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Basic information

Entry
Database: EMDB / ID: EMD-26477
TitleVchTnsC AAA+ with DNA (double heptamer)
Map datapost-processed masked map
Sample
  • Complex: VchTnsC AAA+ with DNA (double heptamer)
    • Protein or peptide: VchTnsC
    • DNA: DNA (36-MER)
    • DNA: DNA (36-MER)
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE
Biological speciesVibrio cholerae (bacteria) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.9 Å
AuthorsFernandez IS / Sternberg SH
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)DP2hG011650-01 United States
CitationJournal: Nature / Year: 2022
Title: Selective TnsC recruitment enhances the fidelity of RNA-guided transposition.
Authors: Florian T Hoffmann / Minjoo Kim / Leslie Y Beh / Jing Wang / Phuc Leo H Vo / Diego R Gelsinger / Jerrin Thomas George / Christopher Acree / Jason T Mohabir / Israel S Fernández / Samuel H Sternberg /
Abstract: Bacterial transposons are pervasive mobile genetic elements that use distinct DNA-binding proteins for horizontal transmission. For example, Escherichia coli Tn7 homes to a specific attachment site ...Bacterial transposons are pervasive mobile genetic elements that use distinct DNA-binding proteins for horizontal transmission. For example, Escherichia coli Tn7 homes to a specific attachment site using TnsD, whereas CRISPR-associated transposons use type I or type V Cas effectors to insert downstream of target sites specified by guide RNAs. Despite this targeting diversity, transposition invariably requires TnsB, a DDE-family transposase that catalyses DNA excision and insertion, and TnsC, a AAA+ ATPase that is thought to communicate between transposase and targeting proteins. How TnsC mediates this communication and thereby regulates transposition fidelity has remained unclear. Here we use chromatin immunoprecipitation with sequencing to monitor in vivo formation of the type I-F RNA-guided transpososome, enabling us to resolve distinct protein recruitment events before integration. DNA targeting by the TniQ-Cascade complex is surprisingly promiscuous-hundreds of genomic off-target sites are sampled, but only a subset of those sites is licensed for TnsC and TnsB recruitment, revealing a crucial proofreading checkpoint. To advance the mechanistic understanding of interactions responsible for transpososome assembly, we determined structures of TnsC using cryogenic electron microscopy and found that ATP binding drives the formation of heptameric rings that thread DNA through the central pore, thereby positioning the substrate for downstream integration. Collectively, our results highlight the molecular specificity imparted by consecutive factor binding to genomic target sites during RNA-guided transposition, and provide a structural roadmap to guide future engineering efforts.
History
DepositionMar 22, 2022-
Header (metadata) releaseJun 8, 2022-
Map releaseJun 8, 2022-
UpdateSep 21, 2022-
Current statusSep 21, 2022Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_26477.png

Downloads & links

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Map

FileDownload / File: emd_26477.map.gz / Format: CCP4 / Size: 40.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationpost-processed masked map
Voxel sizeX=Y=Z: 1.509 Å
Density
Contour LevelBy AUTHOR: 0.15
Minimum - Maximum-0.0018295422 - 2.1613276
Average (Standard dev.)0.0024654428 (±0.035322595)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions220220220
Spacing220220220
CellA=B=C: 331.97998 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_26477_msk_1.map
Projections & Slices
AxesZYX

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Slices (1/2)
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Additional map: unsharpened map

Fileemd_26477_additional_1.map
Annotationunsharpened map
Projections & Slices
AxesZYX

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Additional map: relion post-processed

Fileemd_26477_additional_2.map
Annotationrelion post-processed
Projections & Slices
AxesZYX

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Half map: half map 2

Fileemd_26477_half_map_1.map
Annotationhalf map 2
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Histogram

Histogram (log scale)

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Half map: half map 2

Fileemd_26477_half_map_2.map
Annotationhalf map 2
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Sample components

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Entire : VchTnsC AAA+ with DNA (double heptamer)

EntireName: VchTnsC AAA+ with DNA (double heptamer)
Components
  • Complex: VchTnsC AAA+ with DNA (double heptamer)
    • Protein or peptide: VchTnsC
    • DNA: DNA (36-MER)
    • DNA: DNA (36-MER)
  • Ligand: ADENOSINE-5'-TRIPHOSPHATE

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Supramolecule #1: VchTnsC AAA+ with DNA (double heptamer)

SupramoleculeName: VchTnsC AAA+ with DNA (double heptamer) / type: complex / Chimera: Yes / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Source (natural)Organism: Vibrio cholerae (bacteria)

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Macromolecule #1: VchTnsC

MacromoleculeName: VchTnsC / type: protein_or_peptide / ID: 1 / Number of copies: 14 / Enantiomer: LEVO
Source (natural)Organism: Vibrio cholerae (bacteria)
Molecular weightTheoretical: 35.065273 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: TREARISRAK RAFVSTPSVR KILSYMDRCR DLSDLESEPT CMMVYGASGV GKTTVIKKYL NQAAAAAAAG GDIIPVLHIE LPDNAKPVD AARELLVEMG DPLALYETDL ARLTKRLTEL IPAVGVKLII IDEFQHLVEE RSNRVLTQVG NWLKMILNKT K CPIVIFGM ...String:
TREARISRAK RAFVSTPSVR KILSYMDRCR DLSDLESEPT CMMVYGASGV GKTTVIKKYL NQAAAAAAAG GDIIPVLHIE LPDNAKPVD AARELLVEMG DPLALYETDL ARLTKRLTEL IPAVGVKLII IDEFQHLVEE RSNRVLTQVG NWLKMILNKT K CPIVIFGM PYSKVVLQAN SQLHGRFSIQ VELRPFSYQG GRGVFKTFLE YLDKALPFEK QAGLANESLQ KKLYAFSQGN MR SLRNLIY QASIEAIDNQ HETITEEDFV FASKLTSGDK PNSWKNPFEE GVEVTEDMLR PPPKDIGWED YLRH

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Macromolecule #2: DNA (36-MER)

MacromoleculeName: DNA (36-MER) / type: dna / ID: 2 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 11.05513 KDa
SequenceString:
(DC)(DT)(DC)(DC)(DA)(DG)(DT)(DA)(DC)(DA) (DG)(DC)(DG)(DC)(DG)(DG)(DC)(DT)(DG)(DA) (DA)(DA)(DT)(DC)(DA)(DT)(DC)(DA)(DT) (DT)(DA)(DA)(DA)(DG)(DC)(DG)

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Macromolecule #3: DNA (36-MER)

MacromoleculeName: DNA (36-MER) / type: dna / ID: 3 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 11.099121 KDa
SequenceString:
(DC)(DG)(DC)(DT)(DT)(DT)(DA)(DA)(DT)(DG) (DA)(DT)(DG)(DA)(DT)(DT)(DT)(DC)(DA)(DG) (DC)(DC)(DG)(DC)(DG)(DC)(DT)(DG)(DT) (DA)(DC)(DT)(DG)(DG)(DA)(DG)

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Macromolecule #4: ADENOSINE-5'-TRIPHOSPHATE

MacromoleculeName: ADENOSINE-5'-TRIPHOSPHATE / type: ligand / ID: 4 / Number of copies: 14 / Formula: ATP
Molecular weightTheoretical: 507.181 Da
Chemical component information

ChemComp-ATP:
ADENOSINE-5'-TRIPHOSPHATE / ATP, energy-carrying molecule*YM / Adenosine triphosphate

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7.2
GridModel: Homemade / Material: GOLD / Mesh: 300 / Pretreatment - Type: PLASMA CLEANING
VitrificationCryogen name: NITROGEN

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: OTHER / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.5 µm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 55.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Initial angle assignmentType: ANGULAR RECONSTITUTION
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionAlgorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 125000
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: OTHER
Output model

PDB-7ufm:
VchTnsC AAA+ with DNA (double heptamer)

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